DESCRIPTION: E6-AP (E6-Associated Protein) is a cellular protein that associates with and catalyzes ubiquitination of p53 in anE6-dependent manner. Characterization of E6-AP showed it to be representative of a large family of ubiquitin-protein ligases (or E3 proteins), which have been postulated to play the major role in substrate selectivity. The applicant proposes a model for function of the E6-AP-related E3 proteins which states that they consist of two broad domains: an N-terminal substrate recognition domain, and a C-terminal catalytic domain (the hect domain, for homologous to E6-AP C-terminus) which ubiquitinates bound substrates. The specific aims of this proposal are: 1) to evaluate this model by determining if substrate specificity can be redirected by substitution of N-terminal sequences and if hect domains are functionally interchangeable among E3 proteins, 2) to evaluate this model with respect to E6-AP by precisely mapping the determinants necessary for p53 association and hect domain function, and 3) to identify substrates of Rsp5, an essential yeast hectE3, beginning with predictions of the 2-domain model and taking a combined genetic and biochemical approach. The long-term objectives of this application are to understand the mechanisms of action of the hect E3 proteins and their role, as well as the role of the high-risk HPV E6 proteins (and possible cellular analogs), in directing the specificity of the ubiquitin system. The ultimate goal is a more complete understanding of HPV-associated carcinogenesis and other processes regulated by protein ubiquitination.